The invention relates to chromoionophore comprising an chromophore and an ionophore capable of selectively binding sodium ions for determining sodium ion in a sample. The present invention also relates to a method of determining the concentration of sodium ions in a sample wherein the chromoionophore is contacted with sodium ion in a sample, wherein the intensity of at least one absorption maximum in the visible region changes and the concentration of sodium ion is calculated based on the change in the intensity of the absorption maximum.
The accurate measurement of physiologic cations, such as sodium, potassium, lithium, calcium, and magnesium, is essential in clinical diagnosis. Traditionally, these ions were determined in plasma or serum using ion-selective electrodes (ISE), which are very cumbersome to use and costly to maintain. Serious drawbacks of electrochemical measuring arrangements are the requirement of a reference element, sensitivity towards electrical potentials and electromagnetic interference.
An alternative enzymatic method is based on the activation of β-Galactosidase by cations (Berry et al., Clin. Chem., 34/11, 1988 2295-2298). The high cost and poor stability of the enzyme preclude its extensive application in clinical laboratories. Therefore, the development of practical and inexpensive calorimetric reagents for the clinical determination of these ions in biological fluids remains an important area of research.
U.S. Pat. No. 4,367,072 describes a process for the determination of metal ions using simple crown ethers as ion-binding units. However, the binding is too weak to be useful for many practical applications, such as clinical applications, in which the indicator has to discriminate between ions with very similar properties, e.g., sodium versus potassium or magnesium versus calcium.
U.S. Pat. No. 5,011,924 and U.S. Pat. No. 4,994,395 describe cryptands (or cryptohemispherands) linked with an ionizable chromophore, which changes its color upon binding of ions based on charge interaction between the bound cation and the anion of chromophore. Although all nitrogen atoms in these cryptands are aliphatic, and not electronically conjugated with the chromophore, the results of measurement of serum samples using these chromoionophores are impressive and promising (Helgeson et. al. J. Am. Chem. Soc., vol. 111, 1989, 6339-6350). However, the syntheses of these cryptands, especially of those cryptohemispherands, are lengthy and tedious. Consequently, the manufacturing cost of these reagents remains prohibitively high even in the decades following their discovery. The cost factor could be a reason why these reagents have not replaced those ISE modules in most large clinical analyzers, in which the ISE methods are still dominating (see Burtis et. al. ed. “Tietz Textbook of Clinical chemistry and Molecular Diagnostics” Elsevier Sauders, St. Louis, Mo., USA 2006, page 986).
U.S. Pat. No. 5,952,491 report sodium ionophore, which has π-electron conjugated nitrogen and is coupled to a fluorophore to make luminophore-ionophore sensors where the respective ions are detected by measuring luminescence emission. All three ionophores has been proven to be very effective in determination of sodium in whole blood in which sodium is the major cation. (see He et. al. Anal. Chem. Vol. 75, 2003, 449-555), thus showing that the ionophore is effective under physiological conditions.
By coupling to a chromophoric moiety, the ionophore can be converted into colorimetric sensors. The chromophoric moieties can be a nitro-substituted styryl or phenylazo, substituted thiazolevinyl or thiazoleazo, substituted naphthothiazolevinyl or naphthothiazoleazo, substituted naphthylvinyl or naphthylazo, substituted quinolinovinyl or quinolinoazo and their quartemized salts. To date, there has been no systematic investigation of these types of colorimetric reagents. Gunnlaugsson et al. (J. Chem Soc., Perkin Trans. 2, 2002, 141-150) describe use of a sodium ionophore with a nitrophenylazo chromophore. The water solubility of this dye is so poor that one has to use organic solvent to solubilize it. The water solubility can be improved dramatically if a charge is introduced into the dye molecules. The absorption wavelength can be red-shifted by replacing the nitrophenyl with a nitrothiazole or larger chromophore-generating substituent.
The present invention provides sodium chromoionophores that are water soluble and can be reliably used for detection of ions in samples that absorb at wavelengths longer than about 400 nm. Examples of such samples are biological fluids including plasma, serum and urine.
For the chromoionophores of the present invention, the amount of ion present is determined by measuring changes in the intensity of at least one absorption maximum of the chromoionophore upon contacting the chromoionophore with an ion. The measurements are done by using standard centralized instruments, such as ultraviolet-visible spectrometers. A calibration curve for an ion is generated from a series of empirically determined absorption spectra. A calibration curve is useful for at-once determining the concentration of ion in a sample from the measured absorbance.
The chromoionophores of this invention absorb visible light (about 400 nm or greater) with reasonable extinction coefficient, thus avoiding those practical problems associated with variable background absorption from optical components, cuvette polymer materials, and biological samples. Further, the invention is well suited for practice in the determination of sodium ion in the presence of physiological concentrations of other alkali ions.